Synthesis of oligonucleotides

ABSTRACT

Synthetic processes are provided for the solution phase synthesis of oligonucleotides, especially phosphorothioate oligonucleotides, and intermediate compounds useful in the processes. Intermediates having structure 
                 
 
are prepared in accord with preferred embodiments.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/269,291, filed Oct. 11, 2002 now U.S. Pat. No. 6,646,114, which is acontinuation of U.S. Ser. No. 09/824,474, filed Apr. 2, 2001 now U.S.Pat. No. 6,486,312, which is a continuation of Ser. No. 09/395,948 filedSep. 14, 1999, now U.S. Pat. No. 6,211,350, which is a continuation ofU.S. application Ser. No. 08/692,909 filed Jul. 31, 1996, now U.S. Pat.No. 6,001,982, which is a division of U.S. Ser. No. 08/249,442 filed May26, 1994, now U.S. Pat. No. 5,571,902, which is a continuation-in-partof U.S. Ser. No. 08/099,075 filed Jul. 29, 1993, now U.S. Pat. No.5,614,621, the entirety of which is incorporated herein by reference.

FIELD OF THE INVENTION

This invention is directed to processes for synthesizingoligonucleotides, especially phosphorothioate oligonucleotides, and tointermediates used in that process. This invention is drawn to solutionphase syntheses having improved efficiencies and enhanced convenienceand cost.

BACKGROUND OF THE INVENTION

Oligonucleotides are important materials for research, diagnostic,therapeutic and other purposes. An ever-growing demand for improvedoligonucleotides, oligonucleotide analogs and for methods for theirpreparation and use has arisen. For example, oligonucleotides are widelyused in genomic research as probes, primers and a wide array of otherresearch uses. One widely used technique that uses oligonucleotidesprimers is PCR (polymerase chain reaction) amplification.

Oligonucleotides are also useful in diagnostics since they canspecifically hybridize to nucleic acids of interest in the etiology ofdisease. Oligonucleotides are currently in clinical trials astherapeutic moieties in the treatment of disease states. For example,workers in the field have now identified oligonucleotide compositionsthat are capable of modulating expression of genes implicated in viral,fungal and metabolic diseases. In short, oligonucleotides are importantmolecules having a large commercial impact in biotechnology andmedicine. Improved methods for the synthesis of oligonucleotides are indemand, especially methods which have improvements in cost andconvenience.

The current methods of choice for the preparation of phosphorothioateoligonucleotides employ solid-phase synthesis wherein an oligonucleotideis prepared on a polymer or other solid support. Solid-phase synthesisrelies on sequential addition of nucleotides to one end of a growingoligonucleotide. Typically, a first nucleoside is attached to anappropriate support, e.g. glass, and nucleotide precursors, typicallyphosphoramidites, are added stepwise to elongate the growingoligonucleotide. The nucleotide phosphoramidites are conventionallyreacted with the growing oligonucleotide using the principles of a“fluidized bed” for mixing of the reagents. The silica supports suitablefor anchoring the oligonucleotide are very fragile and thus can not beexposed to aggressive mixing.

In these and other solid-phase procedures the oligonucleotide issynthesized as an elongating strand. However, the number of individualstrands that can be anchored to a unit surface area of the support islimited. Also, the commercially available activated nucleotides that arepresently used to add to a growing oligonucleotide are relativelyexpensive and must be used in stoichiometric excess. Also, theactivating agents, e.g. tetrazole, are used in large excess.

The chemical literature discloses numerous processes for couplingnucleosides through phosphorous-containing covalent linkages to produceoligonucleotides of defined sequence. One of the most popular processesis the phosphoramidite technique (see, e.g., Beaucage, et al.,Tetrahedron 1992, 48, 2223 and references cited therein), wherein anucleoside or oligonucleotide having a free hydroxyl group is reactedwith a protected cyanoethyl phosphoramidite monomer in the presence of aweak acid to form a phosphite-linked structure. Oxidation of thephosphite linkage followed by hydrolysis of the cyanoethyl group yieldsthe desired phosphodiester or phosphorothioate linkage.

The phosphoramidite technique, however, is not without itsdisadvantages. For example, cyanoethyl phosphoramidite monomer is quiteexpensive. Although considerable quantities of monomer go unreacted in atypical phosphoramidite coupling, unreacted monomer can be recovered, ifat all, only with great difficulty. Also, acrylonitrile, the by-productof deprotection of the cyanoethoxy group on the phosphate group iscarcinogenic and in some cases acts as a Michael acceptor to formundesired side-products.

Other exemplary solid state synthetic schemes are set forth in U.S. Pat.No. RE. 34,069 Koster et al.; and U.S. Pat. No. 5,132,418 Carruthers etal.

While currently utilized solid phase syntheses are useful for preparingsmall quantities of oligonucleotide, they typically are not amenable tothe preparation of large quantities of oligonucleotides necessary forbiophysical studies, pre-clinical and clinical trials and commercialproduction. Moreover, such synthetic procedures are very expensive.Thus, although there is a great demand for oligonucleotides, especiallyphosphorothioate oligonucleotides, the art suggests no large scaletechniques for their preparation. Accordingly, there remains a long-feltneed for such methods and for intermediates useful in such methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a reaction schematic depicting exemplary routes for thesyntheses of intermediates useful in the practice of the invention.

FIG. 2 is a schematic showing exemplary coupling of the intermediates ofFIG. 1.

FIG. 3 depicts the coupling of the moieties of FIG. 2.

SUMMARY OF THE INVENTION

In accordance with the invention, methods are provided for the synthesisof oligonucleotide moieties. A first synthon, having the structure

is caused to react with a second synthon having the structure

to form a moiety having the structure

Wherein each Q is independently O, S, CH₂,CHF or CF₂. In accordance withembodiments of the invention, each B_(x) is independently a nucleosidicbase; each X is independently, OH, SH, SCH₃, F, OCN, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃ where n is from 1 to about 10; C₁ to C₁₀ lower alkyl,substituted lower alkyl, alkaryl or aralkyl; Cl, Br, CN, CF₃, OCF₃, O—,S—, or N-alkyl; O—, S—, or N-alkenyl; SOCH₃, SO₂CH₃; ONO₂; NO₂; N₃; NH₂;heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino;substituted silyl; an RNA cleaving group; a conjugate; a reporter group;an intercalator; a group for improving the pharmacokinetic properties ofan oligonucleotide; or a group for improving the pharmacodynamicproperties of an oligonucleotide; Y is a 5′ hydroxyl protecting group; Wis a 3′ hydroxyl protecting group; each Z is O or S; T is a phosphorousblocking group; U is a phosphite activating group; and n is an integerfrom 0 to 50.

In accordance with preferred embodiments, each group T can be asilylalkoxy group wherein the Si atom of this group includes threesubstitutent R groups thereon. Preferably these R groups, independently,are alkyl or aryl with methyl, t-butyl and phenyl being most preferred.U can be a dialkylamino group. In another preferred embodiment, thefirst synthon is formed by coupling of a 3′ hydroxyl protectednucleoside with a 5′ protected nucleoside through a phosphite linkage.

In yet other preferred embodiments, the processes of the invention areaccomplished such that the second synthon is prepared via reaction of aprecursor nucleoside with a reagent [R₁R₂N]₂PO(CH₂)_(x)SiR₃R₄R₅ whereinR₁ and R₂ independently are alkyl having 1 to about 10 carbon atoms, R₃,R₄, and R₅ are, independently, alkyl having 1 to about 10 carbon atomsor aryl having 6 to about 10 carbons atoms, and x is 1 to about 7.Useful as the precursor nucleoside for the preparation of such a secondsynthon is a nucleoside that corresponds in structure to the secondsynthon above, except it bears a hydrogen atom on it 3′ hydroxyl groupin place of the U-P-T groups. Preferably this reaction is conducted inthe presence of 1H-Tetrazole, 5-(4-Nitrophenyl)-1H-tetrazole, ordiisopropylammonium tetrazolide.

It is also preferred that the processes further comprise removing thegroups W, T, and Y from the moiety and oxidizing the moiety to formeither phosphorothioate or phosphodiester bonds.

The processes of the invention may be performed iteratively such thatthe product resulting from a first reaction sequence is transformed intoa “new” first synthon for iterative reaction with a further secondsynthon by removal of the Y group. The processes of the invention myfurther be preformed iteratively such that the product resulting from afirst reaction sequence is transformed into a “new” second synthon foriterative reaction with a further first synthon by removal of the Wgroup followed by phosphitation. While solution phase reactions arepreferred in the practice of this invention, in iterative processes andotherwise, one of the “new” second synthons of the above process, asdescribed in the preceding sentence, can be used in the place of astandard mono-nucleotide phosphoramidite synthon in a standard solidphase reaction to elongated an growing oligonucleotide on a solid phasesupport. Thus instead of adding just a single nucleotide at a time as ispracticed in current solid phase synthesis technology, dimers, trimers,tetramers and even high homologues can be advantageously linked to agrowing oligonucleotide on a solid support. Such dimers, trimers,tetramers and high homologues are prepared using the processes of theinvention and then are carried over and used in place of the standardphosphoamidites of currently practiced solid phase oligonucleotidesynthesis.

In accordance with other embodiments of the invention, there are reactedtogether, in solution, a first synthon comprising at least twonucleoside units having a 5′ location protected with a 5′ hydroxylicblocking group and a 3′ location substituted with a function having theformulaU-P-Twherein U is a phosphite activating group and T is a phosphorousblocking group, with a second synthon comprising a nucleoside unithaving a 3′ location protected with a 3′ hydroxylic blocking group and a5′ location capable of reacting with the U-P-T function. It is preferredthat the product of the reaction be oxidized to form eitherphosphodiester or phosphorothioate internucleoside bonds. It is alsopreferred for some embodiments that the second synthon comprise at leasttwo nucleoside units.

In other preferred methodologies, the function U-P-T is incorporatedthrough reaction with a reagent [R₁R₂N]₂PO(CH₂)_(x)SiR₃R₄R₅ wherein R₁and R₂ independently are alkyl having 1 to about 10 carbon atoms, R₃,R₄, and R₅ are, independently, alkyl having 1 to about 10 carbon atomsor aryl having 6 to about 10 carbons atoms, and x is 1 to about 7.

In other aspects of the invention, compounds are provided having theformulas

Where each Q is, independently O, S, CH₂, CHF or CF₂. In such compounds,each B_(x) is independently a nucleosidic base; each X is independently,OH, SH, SCH₃, F, OCN, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃ where n is from 1 toabout 10; C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl oraralkyl; Cl, Br, CN, CF₃, OCF₃, O—, S—, or N-alkyl; O—, S—, orN-alkenyl; SOCH₃, SO₂CH₃; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a conjugate; a reporter group; an intercalator; agroup for improving the pharmacokinetic properties of anoligonucleotide; or a group for improving the pharmacodynamic propertiesof an oligonucleotide; each Z is independently O or S; Y is H or ahydroxyl protecting group; U is a phosphite activating group; each T isindependently a phosphorous blocking group; and n is an integer from 1to 50.

In accordance with other embodiments of the invention, the phosphorousblocking group has the formula —O(CH₂)_(x)SiR₃R₄R₅ wherein R₃, R₄, andR₅ are, independently, alkyl having 1 to about 10 carbon atoms or arylhaving 6 to about 10 carbons atoms, and x is 1 to about 7.

It is a significant aspect of the present invention that libraries ofoligomers can be constructed and stored to facilitate the synthesis andidentification of useful oligonucleotides. Accordingly, the invention isdirected to libraries comprising a plurality of compounds in accordancewith the foregoing formulas and to oligonucleotides synthesizedtherefrom.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

This invention provides new and improved processes for the preparationof phosphorous-containing covalent linkages and intermediates useful insuch processes. Utilizing these processes and intermediates,phosphodiester and phosphorothioate oligonucleotides are prepared from aplurality of nucleoside or oligonucleotide subunits. The nucleosidesubunits may be “natural” or “synthetic” moieties. Thus, in the contextof this invention, the term “oligonucleotide” in a first instance refersto a polynucleotide formed from a plurality of linked nucleoside units.The nucleosides are formed from naturally occurring bases andpentofuranosyl sugar groups. The term “oligonucleotide” thus effectivelyincludes naturally occurring species or synthetic species formed fromnaturally occurring subunits.

The present invention provides processes for the efficient synthesis ofthe oligonucleotides and modified oligonucleotide compounds (analogs).In preferred embodiments, certain products of the invention are preparedby processes that comprise contacting a protected nucleoside havingformula I, i.e. a precursor nucleoside, with a phosphitylation reagenthaving the formula (R₁R₂N)₂PO(CH₂)_(x)SiR₃R₄R₅ for a time and underreaction conditions effective to form a silylalkyl phosphoramiditemonomer having formula II

wherein:

each Q is, independently, O, S, CH₂, CHF or CF₂;

each B_(x) is, independently, a nucleosidic base;

each X is, independently, OH, SH, SCH₃, F, OCN, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃ where n is from 1 to about 10; C₁ to C₁₀ lower alkyl,substituted lower alkyl, alkaryl or aralkyl; Cl, Br, CN, CF₃, OCF₃, O—,S—, or N-alkyl; O—, S—, or N-alkenyl; SOCH₃, SO₂CH₃; ONO₂; NO₂; N₃; NH₂;heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino;substituted silyl; an RNA cleaving group; a conjugate; a reporter group;an intercalator; a group for improving the pharmacokinetic properties ofan oligonucleotide; or a group for improving the pharmacodynamicproperties of an oligonucleotide;

Y is a 5′ hydroxyl protecting group;

R₁ and R₂, independently, are alkyl having 1 to about 10 carbon atoms;

R₃, R₄, and R₅ are, independently, alkyl having 1 to about 10 carbonatoms or aryl having 6 to about 10 carbons atoms; and

x is 1 to about 7.

Monomers of formula II then are contacted with a 3′ hydroxyl protectednucleoside having formula III for a time and under reaction conditionseffective to form dimers having formula IV

where W is a 3′ hydroxyl protecting group and Y is a 5′ hydroxylprotecting group and R₃, R₄ and R₅ are defined as above.

A dimer having formula IV is then contacted with an oxidizing agent fora time and under reaction conditions effective to form oxidationproducts having formula V (where Z=O or S and n=1). The dimer of formulaV can be converted into a dimeric synthon analogous to either themonomeric synthon of structure II or to the monomeric synthon ofstructure III. Alternatively subsequently reaction of the dimericcompound of structure V with fluoride ion in the conventional mannergives either a phosphodieser or phosphorothioate moiety having formulaVI.

For conversion of the dimer of formula V (where n is 1) to a dimericsynthon analogous to that of structure III above, the 5′ hydroxyblocking group Y is removed in a conventional manner to yield a compoundof formula VII. This compound is then subsequently contacted with afurther phosphitylated nucleoside of formula II under reactionconditions effective to form trimers of formula VIII.

Trimers having formula VIII are then contacted with an oxidizing agentfor a time and under reaction conditions effective to form oxidationproducts having formula v (where n is increased by one). If a trimericproduct is desired, the trimer of formual V is deblocked such as withthe conventional fluoride ion or by the use of tetrafluorosilane in a“wet” solvent to produce either a phosphodiester or phorphorothioatemoiety having formula VI. If higher homologues are desired, the trimerof formula VIII can be deblocked at the 5′ position removing the Yhydroxyl protecting group to yield a synthon analogues to formula VII ordeblocked at the 3′ position removing the W hydroxyl protecting groupand phosphitylated to yield a timeric synthon analogous to the compoundof formula II. Thus a library of building blocks of various lengths canbe formed. Units can be added together as monomers, as dimers, astrimers, etc, for the preparation of higher homologues.

In other embodiments of the present invention, compounds of formula Iare contacted with other suitable coupling reagents to form monomers offormula IIa, wherein U is a phosphite activating group, and T is aphosphorous blocking group. Monomers of formula IIa then are contactedwith 3′ hydroxyl protected nucleoside having formula III for a time andunder reaction conditions effective to form dimers having formula IVa.

where W is a 3″ hydroxyl protecting group, Y is a 5′ hydroxyl protectinggroup and T is a phosphorous protecting group.

A dimer having formula IVa is then contacted with an oxidizing agent fora time and under reaction conditions effective to form oxidationproducts having formula Va (where Z=O or S and n=1).

A dimer of formula Va can also be reacted conventionally to remove 5′hydroxy blocking group Y to yield a compound of formula VIIa (where nis 1) and subsequently contacted with a further phosphitylatednucleoside of formula II under reaction conditions effective to formtrimers of formula VIIIa.

Trimers having formula VIIIa are then contacted with an oxidizing agentfor a time and under reaction conditions effective to form oxidationproducts having formula Va (where n is increased by one). This cycle isrepeated to give the length of the desired product. If such product isthe final product it is subsequently reacted such as with theconventional fluoride ion or tetrafluorosilane in a “wet” solvent toproduce either a phosphodiester or phosphorothioate moiety havingformula VI.

Oligonucleotides of formula Va can also be converted to higher analoguescorresponding to phosphitylated nucleosides of formula II, above. Toform such compounds a dimer of formula Va (n=1) is deprotected at its 3′hydroxyl by removal of the group W and phosphitylated to yield a dimerof formula Vb. The compound of formula Vb can be reacted with a compoundof formula VIIa to form a new compound Vc, having one phosphite linkinggroup located between the portion obtained from the compound of formulaVb and the portion obtained from the compound of formula VIIa.

Subsequent oxidization of the phosphite linkage in Vc to a phosphatelinkages yields a new compound of formula Va where n is the total of then units from both the compound of formula VIIa and the compound offormula Vb.

Oligonucleotides according to the invention also can include modifiedsubunits. Representative modifications include modification of aheterocyclic base portion of a nucleoside or a sugar portion of anucleoside. Exemplary modifications are disclosed in the following U.S.Pat. application Ser. No. 463,358, filed Jan. 11, 1990, entitled“Compositions And Methods For Detecting And Modulating RNA Activity;Ser. No. 566,977, filed Aug. 13, 1990, entitled “Sugar Modifiedoligonucleotides That Detect And Modulate Gene Expression; Ser. No.558,663, filed Jul. 27, 1990, entitled “Novel Polyamine ConjugatedOligonucleotides”; Ser. No. 558,806, filed Jul. 27, 1991, entitled“Nuclease Resistant Pyrimidine Modified Oligonucleotides That Detect AndModulate Gene Expression”; and Serial No. PCT/US91/00243, filed Jan. 11,1991, entitled “Compositions and Methods For Detecting And ModulatingRNA Activity”. Each of these patent applications are assigned to theassignee of this invention. The disclosure of each is incorporatedherein by reference.

The term oligonucleotide thus refers to structures that include modifiedportions, be they modified sugar moieties or modified base moieties,that function similarly to natural bases and natural sugars.Representative modified bases include deaza or aza purines andpyrimidine used in place of natural purine and pyrimidine bases;pyrimidine having substituent groups at the 5 or 6 position; purineshaving altered or replacement substituent groups at the 2, 6, or 8positions. Representative modified sugars include carbocyclic or acyclicsugars, sugars having substituent groups at their 2′ position, andsugars having substituents in place of one or more hydrogen atoms of thesugar. Other altered base moieties and altered sugar moieties aredisclosed in U.S. Pat. No. 3,687,808 and PCT application PCT/US89/02323.

In certain embodiments, the compounds of the invention include conjugategroups. Conjugate groups of the invention include intercalators,reporter molecules, polyamines, polyamides, polyethylene glycols,polyethers, groups that enhance the pharmacodynamic properties ofoligomers, and groups that enhance the pharmacokinetic properties ofoligomers. Typical conjugate groups include cholesterols, phospholipids,biotin, phenanthroline, phenazine, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups thatenhance the pharmacodynamic properties, in the context of thisinvention, include groups that improve oligomer uptake, enhance oligomerresistance to degradation, and/or strengthen sequence-specifichybridization with RNA. Groups that enhance the pharmacokineticproperties, in the context of this invention, include groups thatimprove oligomer uptake, distribution, metabolism or excretion.Representative conjugate groups are disclosed in International PatentApplication PCT/US92/09196, filed Oct. 23, 1992, U.S. patent applicationSer. No. 116,801, filed Sep. 3, 1993, and U.S. Pat. No. 5,218,105. Eachof the foregoing is commonly assigned with this application. The entiredisclosure of each is incorporated herein by reference.

Altered base moieties or altered sugar moieties also include othermodifications consistent with the spirit of this invention. Sucholigonucleosides are best described as moieties that are structurallydistinguishable from yet functionally interchangeable with naturallyoccurring or synthetic wild type oligonucleotides. All sucholigonucleotides are comprehended by this invention so long as theyfunction effectively to mimic the structure of a desired RNA or DNAstrand.

Oligonucleotides prepared via the process and intermediates of theinvention can be used as primers for enzymes which polymerizenucleotides, for example, polymerases and reverse transcriptases, suchas in the well-known PCR technology. Oligonucleotides can also be usedas probes for specific oligonucleotide sequences. Conventional methodsfor use of ologonucleotides as probes are disclosed in, for example,“Molecular Cloning; A Laboratory Manual” 2d. Ed., J. Sambrook et al.,Cold Spring Harbor Press, at chapter 10 and the references therein,which are hereby incorporated by reference.

While compounds of the invention as small as dimeric units are useful,preferred the olignucleotides of the invention comprise from about 10 toabout 30 subunits, although oligomers of up to 50 or even severalhundred subunits may be useful. It is more preferred that sucholigonucleotides comprise from about 15 to 25 subunits. As will beappreciated, a subunit is a base and sugar combination suitably bound toadjacent subunits through a phosphorous-containing linkage. When used as“building blocks” in assembling oligonucleotides, even smallerassemblies are preferred. The smallest, a dinucleotide assembly, is twonucleotide linked by a protected phosphorothioate or phosphodiesterlinkage.

One use of oligonucleotides is in targeting RNA or DNA. One specificsuch use is targeting of RNA or DNA that can code for protein. It ispreferred that the RNA or DNA portion which is targeted using theoligonucleotide be preselected to comprise that portion of DNA or RNAwhich codes for the protein whose formation or activity is to bemodulated. The targeting portion of the composition to be employed is,thus, selected to be complementary to the preselected portion of DNA orRNA. Such use that is to be an antisense oligonucleotide for thatportion.

In accordance with one preferred embodiment of this invention, acompound described as Oligonucleotide 5320 having the structure T₂G₄T₂can be prepared using the process and intermediates of the invention.The synthesis of this compound is shown in the FIGS. 1, 2 and 3 of thisspecification. As is described in the “Abstract” section of Wyatt, etal., Proc. Natl. Acad. Sci., 1994, 91, 1356-1360, “The phosphorothioateoligonucleotide T₂G₄T₂ was identified as an inhibitor of HIV infectionin vitro . . . ”

Other preferred compounds having complementary sequences for herpes,papilloma and other viruses can also be prepared utilizing the processof the invention. Oligonucleotides are also used as research reagents.For example, phosphorothioate oligonucleotides have been utilized in thestudies of enzyme biochemistry and protein-nucleic acid interactions.These and other applications of phosphorothioate oligonucleotides arelisted in “Oligonucleotides and Analogues A Practical Approach”, F.Eckstein Ed., IRL Press, at pages 88-91, and in the references containedtherein, which are hereby incorporated by reference.

In one aspect, the present invention is directed to synthetic methodswherein a protected nucleoside having formula I is contacted with acoupling reagent such as one having the formula(R₁R₂N)₂PO(CH₂)_(x)SiR₃R₄R₅ for a time and under conditions effective toform a silylalkyl phosphoramidite monomer having formula II.

Such contacting preferably is effected under anhydrous conditions in thepresence of a weak acid like 1H-tetrazole or5-(4-nitrophenyl)-1H-tetrazole or diisopropylammonium tetrazolide or anyother acid.

Q can be S, CH₂, CHF CF₂ or, preferably, O. In a further preferred groupof compounds, Q is S (see, e.g., Secrist, et al., Abstract 21, Synthesisand Biological Activity of 4′-Thionucleosides, Program & Abstracts,Tenth International Roundtable, Nucleosides, Nucleotides and theirBiological Applications, Park City, Utah, Sept. 16-20, 1992). Each Q isindependently selected and, hence, can be the same as or different fromother Q within a given compound.

B_(x) can be nucleosidic bases selected from the natural nucleosidicbases adenine, guanine, uracil, thymine, cytosine as well as other basesincluding 2-aminoadenosine or 5-methylcytosine. In addition, othernon-naturally occurring species can be employed to provide stable duplexor triplex formation with, for example, DNA. Representative bases aredisclosed in U.S. Pat. No. 3,687,808 (Merigan et al.) which isincorporated herein by reference.

X can be OH, SH, SCH₃, F, OCN, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃ where n isfrom 1 to about 10; C₁ to C₁₀ lower alkyl, substituted lower alkyl,alkaryl or aralkyl; Cl, Br, CN, CF₃, OCF₃, O—, S—, or N-alkyl; O—, S—,or N-alkenyl; SOCH₃, SO₂CH₃; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a conjugate; a reporter group; an intercalator; agroup for improving the pharmacokinetic properties of anoligonucleotide; or a group for improving the pharmacodynamic propertiesof an oligonucleotide. It is intended that the term “alkyl” denotebranched and straight chain hydrocarbon residues, including alkyl groupshaving one or more ³H and/or ¹⁴C atoms. It is preferred that X is R orOH, or, alternatively F, O-alkyl or O-alkenyl, especially where Q is O.Preferred alkyl and alkenyl groups have from 1 to about 10 carbon atoms.

Representative 2′-O-substituted oligonucleotides are described in PCTpatent application WO 93/13121, filed Dec. 23, 1992, entitled Gapped 2′Modified Phosphorothioate Oligonucleotides, and U.S. patent applicationSer. No. US93/06807 filed Jul. 20, 1993 entitled Novel 2′-O-AlkylNucleosides and Phosphoramidites Processes for the Preparation and UsesThereof and in the references within these applications. Bothapplications are assigned to the same assignee as this application, andare hereby incorporated by reference.

For example, in the above cited references 2′-O-substituted nucleosidesare disclosed, including 2′-O-alkyl (methyl through octadecyl); -allyl,-dimethylallyl, -(N-phthalimido)prop-3-yl and -(N-phthalimido)pent-3-yl; -(imidazol-1-yl)butyl; and -pentyl-w-(N-phthalimido). Othersubstitutent groups disclosed in the references as useful forsubstitution at the 2′ position of nucleosides include fluoro, C1-C9aminoalkoxy; C1-C9 alkylimidazole, and polyethyleneglycol.

Y can be any hydroxyl protecting group for the 5′ hydroxyl function ofnucleosides. Preferably, Y is stable under basic conditions but can beremoved under acidic conditions. A wide variety of protecting groups canbe employed in the methods of the invention. In general, protectinggroups render chemical functionality inert to specific reactionconditions, and can be appended to and removed from such functionalityin a molecule. Representative protecting groups are disclosed byBeaucage, S. L.; Uyer, R. P., Advances in the Synthesis ofOligonucleotides by the Phosphoramidite Approach, Tetrahedron, 1992, 48,2223-2311. Preferred protecting groups include dimethoxytrityl (DMTr),monomethoxytrityl, 9-phenylxanthen-9-yl (Pixyl) and9-(p-methoxyphenyl)xanthen-9-yl (Mox).

Coupling reagents preferably have the formula(R₁R₂N)₂PO(CH₂)_(x)SiR₃R₄R₅ where R₁ and R₂ independently are alkylhaving 1 to about 10 carbon atoms, R₃, R₄, and R₅ are, independently,alkyl having 1 to about 10 carbon atoms or aryl having 6 to about 10carbons atoms, and x is 1 to about 7. These are preferably prepared byreacting an alcohol having formula HO(CH₂)_(x)SiR₃R₄R₅ with phosphorustrichloride and reacting the resultant product, CL₂PO(CH₂)_(x)SiR₃R₄R₅,with at least two equivalents of an amine having formula R₁R₂NH. Theammoniacal R groups can be the same or different and may be alkyl having1 to about 10 carbon atoms, preferably 1 to 6 carbon atoms, morepreferably 3 carbon atoms. Isopropyl groups are particularly preferred.The silyl R groups can be the same or different and may be alkyl having1 to about 10 carbon atoms or aryl having 6 to about 10 carbon atoms.Preferably, R₃, R₄ and R₅ are selected from ethyl, ethyl, isopropyl,propenyl, n-butyl, t-butyl, and phenyl groups. Preferably, two of thesesilyl R groups are phenyl groups and one is a methyl group.

In the formula of the above coupling groups, the variable x can be 1 toabout 7, preferably 1 to about 4, more preferably 2. A number ofsuitable alcohols are disclosed by U.S. Pat. No. 5,159,095, issued Oct.27, 1992 in the name of Celebuski, which is incorporated herein byreference. One preferred coupling reagent is diphenylmethylsilylethylN,N-diispropylphosphoramidite, which can be derived fromdiphenylmethylsilylethyl alcohol via diphenylmethylsilylethylphosphodichloridite.

Silylalkyl phosphoramidite monomers having formula II can be contactedwith 3′-hydroxyl protected nucleosides having formula III for a time andunder conditions effective to form phosphite dimers having formula IV,where the group w is a 3′ hydroxyl protecting group.

In preferred embodiments, such contact is effected under anhydrousconditions in the presence of an activating agent such as 1H-tetrazole,5-(4-nitrophenyl)-1H-tetrazole, or diisopropylammonium tetrazolide.

W can be any hydroxyl protecting group for the 3′ hydroxyl function ofnucleosides. Preferably, W is stable under acidic conditions but can beremoved under basic conditions. A wide variety of protecting groups canbe employed in the methods of the invention. Preferred protecting groupsinclude acyl protecting groups. A particularly preferred acyl group isacetyl. Other useful blocking groups that are stable under acidconditions but that can otherwise be removed are as is described inBeaucage et al., ibid.

Protecting groups for the compounds of the invention are selected suchthat the 3′-hydroxyl terminus protecting group W and the 5′-hydroxylterminus protecting group Y are removed under different conditions.Protecting groups for the phosphite moiety, for example the—O(CH₂)xSiR₃R₄R₅ group of compounds of formula II, are chosen such thatthey are stable to conditions required for the removal of either the3′-hydroxyl terminus protecting group w or the 5′-hydroxyl terminusprotecting group Y. Protecting groups for nucleobases are chosen suchthat they are stable to conditions required for the removal of either3′-hydroxyl terminus protecting group W or 5′-hydroxyl terminusprotecting group Y.

Phosphite compounds having formula IV can then be oxidized to producecompounds having formula V (n=1).

Such oxidation can be effected to both phosphodiester (Z=O) andphosphorothioate (Z=S) structures. Useful sulfurizing agents includeBeaucage reagent described in e.g., Iyer, R. P.; Egan, W.; Regan, J. B.;Beaucage, S. L., 3H-1,2-Benzodithiole-3-one 1,1-Dioxide as an ImprovedSulfurizing Reagent in the Solid-Phase Synthesis ofOligodeoxyribonucleoside Phosphorothioates, Journal of American ChemicalSociety, 1990, 112, 1253-1254 and Iyer, R. P.; Phillips, L. R.; Egan,W.; Regan J. B.; Beaucage, S. L., The Automated Synthesis ofSulfur-Containing Oligodeoxyribonucleotides Using3H-2-Benzodithiol-3-one 1,1-Dioxide as a Sulfur-Transfer Reagent,Journal of Organic Chemistry, 1990, 55, 4693-4699. Tetraethyl-thiuramdisulfide can also be used as described in Vu, H.; Hirschbein, B. L.,Internucleotide Phosphite Sulfurization With TetraethylthiuramDisulfide, Phosphorothioate Oligonucleotide Synthesis ViaPhosphoramidite Chemistry, Tetrahedron Letters, 1991, 32, 3005-3007.Further useful reagents for this step are dibenzoyl Tetrasulfide, Rao,M. V.; Reese, C. B.; Zhengyun, Z., Dibenzoyl Tetrasulphide—A RapidSulphur Transfer Agent in the Synthesis of Phosphorthioate Analogues ofOligonucleotides, Tetrahedron Letters, 1992, 33, 4839-4842;di(phenylacetyl)disulfide, Kamer, R. C. R.; Roelen, H. C. P. F.; van denEist, H.; van der Marel, G. A.; van Boom, J. H., An Efficient ApproachToward the Synthesis of Phosphorothioate Diesters Va the SchonbergReaction, Tetrahedron Letters, 1989, 30, 6757-6760; sulfur; and sulfurin combination with ligands like triaryl, trialkyl or triaralkyl ortrialkaryl phosphines. Useful oxidizing agents includeiodine/tetrahydrofuran/water/pyridine, hydrogen peroxide/water,tert-butyl hydroperoxide, or a peracid like m-chloroperbenzoic acid. Inthe case of sulfurization, reaction is performed under anhydrousconditions with the exclusion of air, in particular oxygen, whereas inthe case of oxidation the reaction can be performed under aqueousconditions.

In certain embodiments, compounds having formula V are exposed toreaction conditions effective to remove the 5′ hydroxyl protecting groupY, and the resultant product is contacted with additional monomer II toform phosphite oligonucleotides having formula VIII (n=1).

As will be recognized, further compounds having formula VIII, wherein Yand W are hydroxyl protecting groups and n is, for example, 2-200, canbe prepared by oxidizing the phosphite intermediate to the phosphate,followed by removing the Y hydroxyl protecting group, and reacting withadditional monomer II. It will further be recognized, that multipleiteration of this reaction can be effect to extend the oligonucleotidefor increasing values of n.

In preferred embodiments, compound having formula IV are exposed toreaction conditions effective to remove the 5′ hydroxyl protecting groupY, and reacting the resultant product is with additional monomer II toform phosphite oligonucleotides having formula IVb (n=2).

As will be recognized, further compounds having formula IVb, wherein Wis a hydroxyl protecting group and n is, for example, 3-200, can beprepared by removing the 5′-hydroxyl protecting group Y and addingadditional monomer of formula II.

Oxidation products having, for example, formula V can be exposed toconditions effective to cleave the 3′-OH protecting group W. Theresulting product is contacted with ammonium hydroxide or some otheraqueous base or fluoride ion to remove the silylalkyl portion thereof toyield phosphodiester and phosphorothioate-containing compounds having,for example, formula IX.

Contact with fluoride ion preferably is effected in a solvent such astetrahydrofuran or acetonitrile or dimethoxyethane or water. Fluorideion preferably is provided in the form of one or more salts selectedfrom tetraalkylammonium fluorides (e.g., tetrabutylammonium fluoride(TBAF) or potassium fluoride or Cesium fluoride.

A more preferred method of removing the phosphorous protecting group iseffected via the use of tetrafluorosilane in a “wet” solvent. Suitableas the solvent is acetonitrile having a trace of water. A 100:1 mixturegave essentially immediate deprotection at room temperature without anyindication of cleavage of the internucleotide linkage. While we do notwish to be bound by theory, it is believed that this deprotection iseffected via a β-fragmentation mechanism that is a result ofcoordination of the silicon atom of the tetrafluorosilane with the etheroxygen atom of the protecting group. Electron withdrawal at the etheroxygen atom is augmented by the electron withdrawing effects of thefluorine atoms of the tetrafluorosilane moiety.

The 3′-hydroxyl protecting group Y can be removed from compounds having,for example, formula IX by techniques well known in the art to producecompounds having formulas VI and VIa wherein Y is replaced by H. Forexample, dimethoxytrityl protecting groups can be removed by proticacids such as formic acid, dichloroacetic acid, trichloroacetic acid,p-toluene sulphonic acid or any other acid or with Lewis acids like zincbromide or any other Lewis acid.

The 3′-hydroxyl protecting group W can be removed from compounds having,for example, formula IX by techniques well known in the art to producecompounds wherein w is replaced by H. For example, acetyl protectinggroups can be removed by barium hydroxide.

In a preferred embodiment, compounds of the present invention arereacted in iterative fashion to produce oligonucleotides of up to about200 units. A more preferred size is up to about 50 units. FIG. 1 is areaction schematic depicting exemplary routes for the preparation ofintermediates useful in the synthesis of oligonucleotides. 3′-O-Acetyldeoxythymidine is reacted with 5′-DMT DPSE phosphoramidite, monomer 2,and subsequently oxidized to form dimer 3, which has hydroxyl protectinggroups at the 3′- and 5′-hydroxyl termini. Dimer 3 is then eitherdeprotected at the 5′-hydroxyl to yield dimer 4, or deprotected at the3′-hydroxyl and phosphitylated to produce activated dimer 5. 3′-O-Acetyldeoxyguanine is reacted with 5′-DMT DPSE phosphoramidite 6 andsubsequently sulfurized, i.e. oxidized, to form dimer 7, which is theneither deprotected at the 5′-hydroxyl to yield dimer 8, or deprotectedat the 3′-hydroxyl and phosphitylated to produce activated dimer 9.

FIG. 2 shows the coupling of dimer 8 and activated dimer 5 andsubsequent sulfurization, 3′-O-deactylation, and phosphitylation toyield activated tetramer 10. FIG. 2 also shows the coupling of dimer 4and activated dimer 9 and subsequent sulfurization and 5′-hydroxyldeprotection to yield 3′-O-acetylated tetramer 11.

FIG. 3 shows the coupling and subsequent sulfurization of3′-O-acetylated tetramer 11 and activated tetramer 10 to yield the fullyprotected octamer 12, and subsequent reaction removal all protectinggroups to yield oligomer T-T-G-G-G-G-T-T. As will be recognized,oligomers containing odd numbers of nucleotide units may be synthesizedby incorporating a monomer of formula II or another nucleosidic moietycontaining an odd number of nucleoside subunits into the iterativeprocess at any point in the process.

It will also be recognized that the processes of the invention may alsobe accomplished by reaction of a nucleotide containing a free hydroxylat the 3′ terminus with a nucleosidic moiety phosphitylated at the5′-hydroxy terminus.

Additionally, it will be recognized that the process steps of thepresent invention need not be performed any particular number of timesor in any particular sequence. Accordingly, products incorporating oneor several functional groupings of the enumerated formulas may beconstructed according to the invention. For example, the iterativeoxidation of a growing oligonucleotide chain may be independentlyachieved with sulfurizing agents or oxidizing agents to yield oligomerscontaining both phosphate and phosphorothioate groups.

It will be further seen that by substitution of the alkylsilyl ethoxygroup of the coupling reagent (R₁R₂N)₂PO(CH₂)_(x)SiR₃R₄R₅ the process ofthe present invention may be employed to make otherphosphorous-substituted oligonuclotides. For example, substitution ofone or more of the alkylsilylethoxy groups with methyl groups wouldresult in a methylphosphite intermediate, capable of subsequentoxidation by the methods of the invention to yield an oligonucleotidecontaining one or more methyl phosphonate linkages. In a like manner,use of β-cyanoethyl phosphorous groups can be employed. Analogously, itmay be seen that starting with a phosphorothioamidite, the process ofthe invention may be utilized to produce oligonucleotides containing oneor more phosphorodithioate linkages.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing example thereof, which are not intended to be limiting.

EXAMPLE 1

Preparation of Fully Protected TT Dimer:

To a solution of 1.42 g (5.0 mmol) of 3′-acetyl thymidine and 0.35 g(4.0 mmol) of 1-H Tetrazole in acetonitrile (60 mL) is added 5.50 /g(6.0 mmol) of 5′-O-(4,4′-dimethoxytrityl)thymidine-3′-O-(2-diphenylmethylsilylethylN,N-diisopropylphosphoramidite in 40 mL acetonitrile. The reactionmixture is stirred at room temperature under argon for 0.5 h. A solutionof 5.0 g (25 mmol) of 3H-1,2-benzodithiol-3-one 1,1-dioxide inacetonitrile is added as quickly as possible with vigorous stirring. Thereaction mixture is stirred at room temperature for 20 minutes. Thereaction mixture is then filtered and concentrated. The crude product ispurified by flash chromatography on silica gel using ethylacetate/hexane. 1% triethylamine is used during the purification.³¹P-NMR (CDCl₃, ppm): 66.80, 67.07.

EXAMPLE 2

Preparation of Fully Protected GG Dimer:

To a solution of 3′-acetyl-N²-isobutyryl-2′-deoxyguanosine (5.0 mmole)and 1-H Tetrazole (4.0 mmole) in acetonitrile (60 mL) is added 5.50 g(6.0 mmol) ofN²-Iso-butyryl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine-3′-O-(2-diphenylmethylsilylethylN,N-diisopropylphosphoramidite) in acetonitrile (40 mL). The reactionmixture is stirred at room temperature under argon for 0.5 h. A solutionof 5.0 g (25 mmole) of 3H-1,2-benzodithiol-3-one 1,1-dioxide inacetonitrile is added as quickly as possible with vigorous stirring. Thereaction mixture is stirred at room temperature for 20 minutes. Thereaction mixture is then filtered and concentrated. The crude product ispurified by flash chromatography on silica gel using ethylacetate/hexane. 1% Triethylamine is used during the purification.³¹P-NMR (CDCl₃, ppm): 66.8, 67.1.

EXAMPLE 3

Preparation of the 5′-HO-TT Dimer:

Fully protected phosphorothioate TT dimer (1 mmol) is dissolved indichloromethane (50 mL) and 3% dichloroacetic acid in dichloromethane(v/v) (20 mL) is added and stirred for 15 minutes. The reaction mixtureis concentrated and purified by flash chromatography on silica gel usingethyl acetate/hexane. 1% Triethylamine is used during the purification.³¹P-NMR (CDCl₃, ppm): 67.3, 67.41.

EXAMPLE 4

Preparation of the 5′-HO-GG Dimer:

Fully protected phosphorothioate GG dimer (1 mmol) is dissolved indichloromethane (50 mL) and 3% dichloroacetic acid in dichloromethane(v/v) (20 mL) is added and stirred for 15 minutes. The reaction mixtureis concentrated and purified by flash chromatography on silica gel usingethyl acetate/hexane. 1% Triethylamine is used during the purification.³¹P-NMR (CDCl₃, ppm): 67.5, 67.6.

EXAMPLE 5

Preparation of Fully Protected TTT Trimer:

To a stirred solution of 5′-HO-TT phosphorothioate dimer (5.0 mmole) and1-H Tetrazole (5.0 mmole) in acetonitrile (60 mL) is added5′-O-(4,4′-dimethoxytrityl) thymidine-3′-O(2-diphenylmethylsilylethylN,N-diisopropylphosphoramidite) (6.0 mmol) in acetonitrile (40 mL). Thereaction mixture is stirred at room temperature under argon for 0.5 h. Asolution of 3H-1,2-benzodithiol-3-one 1,1-dioxide (25 mmol) inacetonitrile is added as quickly as possible with vigorous stirring. Thereaction mixture is stirred at room temperature for 20 minutes. Thereaction mixture is then filtered and concentrated. The crude product ispurified by flash chromatography on silica gel using ethylacetate/hexane. 1% Triethylamine is sued during the purification.

EXAMPLE 6

Preparation of Fully Protected CTT Trimer:

To a solution of 5′-HO-TT phosphorothioate dimer (5.0 mmole) and 1-HTetrazole (5.0 mmole) in acetonitrile (60 mL) is addedN⁴-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine-3′-O-(2-diphenylmethylsilylethylN,N-diisopropylphosphoramidite) (6.0 mmol) in acetonitrile (40 mL). Thereaction mixture is stirred at room temperature under argon for 0.5 h. Asolution of 3H-1,2-benzodithiol-3-one 1,1-dioxide (25 mmol) inacetonitrile is added as quickly as possible with vigorous stirring. Thereaction mixture is stirred at room temperature for 20 minutes. Thereaction mixture is then filtered and concentrated. The crude product ispurified by flash chromatography on silica gel using ethylacetate/hexane. 1% Triethylamine is used during the purification.

Those skilled in the art will appreciate that numerous changes andmodifications may be made to the preferred embodiments of the inventionand that such changes and modifications may be made without departingfrom the spirit of the invention. It is therefore intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the invention.

1. A compound having the formula:

wherein: each Q is independently O, S, CH₂, CHF or CF₂; each Bx isindependently a nucleosidic base; each X is independently, OH, SH, SCH₃,F, OCN, O(CH₂)_(n)NH₂, C₁ to C₁₀ lower alkyl, C₁ to C₁₀ substitutedlower alkyl, alkaryl, aralkyl, Cl, Br, CN, CF₃, OCF₃, O-alkyl, S-alkyl,N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃,NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino, substituted silyl, an RNA cleaving group, a conjugate, areporter group, or an intercalator; W is a 3′ hydroxyl protecting group;each Z is independently O or S; each T is independently a phosphorousblocking group each Y is H or a hydroxyl protecting group; and n is 1,3, 5 or
 7. 2. The compound of claim 1 wherein n is
 1. 3. The compound ofclaim 1 wherein n is
 3. 4. The compound of claim 1 wherein n is
 5. 5.The compound of claim 1 wherein n is
 7. 6. The compound of claim 1wherein X is selected from the group consisting of C₁ to C₁₀ loweralkyl, O-alkyl, S-alkyl, N-alkyl, N₃, NH₂, and OH.
 7. The compound ofclaim 1 wherein X is C₁ to C₁₀ lower alkyl.
 8. The compound of claim 1wherein X is O-alkyl, S-alkyl, or N-allkyl.
 9. The compound of claim 1wherein X is N₃ or NH₂.
 10. The compound of claim 1 wherein X is OH.